1. Field of the Invention
The present invention relates to the compaction of nucleic acids and the delivery of compacted exogenous nucleic acids to cells of multicellular organisms, in vivo.
2. Description of the Background Art
Functional exogenous genes can be introduced to mammalian cells in vitro by a variety of physical methods, including transfection, direct microinjection, electroporation, and coprecipitation with calcium phosphate. Most of these techniques, however, are impractical for delivering genes to cells within intact animals.
Receptor-Mediated Uncompacted DNA Delivery In Vivo.
Receptor-mediated gene transfer has been shown to be successful in introducing transgenes into suitable recipient cells, both in vitro and in vivo. This procedure involves linking the DNA to a polycationic protein (usually poly-L-lysine) containing a covalently attached ligand, which is selected to target a specific receptor on the surface of the tissue of interest. The gene is taken up by the tissue, transported to the nucleus of the cell and expressed for varying times. The overall level of expression of the transgene in the target tissue is dependent on several factors: the stability of the DNA-carrier complex, the presence and number of specific receptors on the surface of the targeted cell, the receptor-carrier ligand interaction, endocytosis and transport of the complex to the nucleus, and the efficiency of gene transcription in the nuclei of the target cells.
Wu, et al., U.S. Pat. No. 5,166,320, discloses tissue-specific delivery of DNA using a conjugate of a polynucleic acid binding agent (such as polylysine, polyarginine, polyornithine, histone, avidin, or protamine) and a tissue receptor-specific protein ligand. For targeting liver cells, Wu suggests "asialoglycoprotein (galactose-terminal) ligands". These may be formed, Wu says, either by desialation of appropriate glycoproteins, or by coupling lactose to non-galactose bearing proteins. The molar ratio of polynucleic acid to conjugate is in the range 1:10 to 10:1, more typically 1:5 to 5:1, more preferably 1:2 to 3:1. While not stated by Wu et al., in our hands, Wu's method resulted in structures with a diameter of at least 80 nm.
Low, et al., U.S. Pat. No. 5,108,921, disclose binding biotin to DNA to transform a cell using receptor mediated endocytosis.
Stomp, et al., U.S. Pat. No. 5,122,466 and McCabe, et al., U.S. Pat. No. 5,120,657 disclose attaching DNA to a metal pellet by covalently attaching polylysine to the material and then allowing DNA to be complexed to it. The resulting product is then used for ballistic transformation of a cell. See Stomp, et al., column 7, lines 29-37 and McCabe, et al., column 7, lines 49-65.
Wagner, et al., Proc. Natl. Acad. Sci., 88:4255-4259 (1991) disclose complexing a transferrin-polylysine conjugate with DNA for delivering DNA to cells via receptor mediated endocytosis. Wagner, et al., teach that it is important that there be sufficient polycation in the mixture to ensure compaction of plasmid DNA into toroidal structures of 80-100 nm diameter, which, they speculate, facilitate the endocytic event. Wagner et al. do not recognize the value of attaining smaller diameter structures or teach how to obtain a greater degree of compaction. It is believed that Wagner et al's structures are multimolecular complexes, which have the disadvantage that they are more vulnerable to macrophage phagocytosis and less amenable to uptake by target tissues.
Direct injection of Naked, Uncompacted DNA. The possibility of detecting gene expression by directly injecting naked DNA into animal tissues was demonstrated first by Dubenski et al, Proc. Nat. Acad. Sci. USA, 81:7529-33 (1984), who showed that viral or plasmid DNA injected into the liver or spleen of mice was expressed at detectable levels. The DNA was precipitated using calcium phosphate and injected together with hyaluronidase and collagenase. The transfected gene was shown to replicate in the liver of the host animal. Benvenisty and Reshef, Proc. Nat. Acad. Sci. USA, 83:9551-55 (1986) injected calcium phosphate precipitated DNA intraperitoneally into newborn rats and noted gene expression in the livers of the animals 48 hr. after transfection. In 1990, Wolff et al, Science, 247:1456-68 (1990), reported that the direct injection of DNA or RNA expression vectors into the muscle of mice resulted in the detectable expression of the genes for periods for up to 2 months. This technique has been extended by Acsadi et al, New Biologist, 3:71-81 (1991) to include direct injection of naked DNA into rat hearts; the injected genes were expressed in the heart of the animals for up to 25 days. Other genes, including the gene for dystrophin have been injected into the muscle of mice using this technique. This procedure forms the base of a broad approach for the generation of immune response in an animal by the administration of a gene by direct injection into the target tissue. The gene is transiently expressed, producing a specific antigen (see Donnelly et al, The Immunologist, 21, pp. 20-26 (1994) for a recent review). However, the DNA used in these experiments has not been modified or compacted to improve its survival in the cell, its uptake into the nucleus or its rate of transcription in the nucleus of the target cells.
Behavior of DNA in Solution.
DNA is a rod-like molecule in solution, due to the highly negatively charged nature of its phosphate backbone, and its basic structure can be perturbed by modification of the hydration shell associated with the helix. This perturbation can be brought about in two ways; first, a change in the degree of charge neutralization of the DNA molecules resulting in extensive compaction and eventually in the separation of the DNA phase (precipitation) in the form of compact structures, and second, a change in the dielectric constant of the DNA helix leading to the formation of compact structures. These perturbations result in a change in the conformation of the DNA molecule permitting the flexible polymer to bend and become compacted, markedly altering the hydrodynamic properties of the DNA molecule. The resultant structures are thought to be of similar nature to that which the DNA assumes in the chromosomes of higher eukaryotes and inside viral capsids.
DNA in the nucleus of a higher eukaryote is intimately associated with basic nuclear proteins (i.e. the histones and protamines) with a high content in lysine and arginine (histones) or arginine (protamines). The complex of DNA with these basic proteins is responsible for the control of DNA compaction that occurs upon chromosome formation and is thought to play a role in the regulation of gene expression. DNA compaction, which occurs physiologically in viruses, bacteria and eukaryote nuclei, has been extremely difficult to reproduce in the laboratory. Theoretically, due to the highly negatively charged nature of the DNA backbone, a change in the degree of charge neutralization of the DNA results in extensive compaction and eventually in the separation of the DNA phase (precipitation) in the form of compact structures. However, the behavior of DNA-polycation complexes in solution is dependent on the method for complexing DNA with the polycationic protein.
Studies by Olins, Olins and von Hipple (J. Mol. Bio. 24, 157-176, 1967) using cationic homopolypeptides as models for nucleoprotein complex formation presented evidence for the formation of specific complexes of DNA with cationic polypeptides (poly-L-lysine, poly-L-arginine and poly-L-ornithine) after "annealing" of both components in solution. This procedure involved step-down dialysis from NaCl concentrations of 2M to 0.010 M.
Several comments may be made on this study. First, thermal denaturation of complexes formed by the addition of polycation to DNA established that polycation binding to DNA occurred in every case studied, and resulted in the stabilization of the double stranded structure of DNA. It is important to note that this system differs from that in which a change in the dielectric constant (i.e. alcohol dehydration) results in DNA collapse with no change in the thermal denaturation characteristics of the DNA. Second, spectrophotometric studies indicated that the absorbance maxima at 260 nm was shifted slightly to the red with a progressive increase in turbidity at wavelengths greater than 300 nm (a region in which neither the polycation nor the DNA show any absorbance). These characteristics were thought to indicate that a small conformational change, occurring possibly through the interaction between DNA and the polypeptide, was being detected by an anomalous absorption spectra. Third, the complexes formed by the addition of basic polypeptides to DNA resulted in molecular aggregation and the formation of precipitates.
Optical Rotatory Dispersion and Circular Dichroism were applied to the study of the interaction between basic homopolypeptides and DNA in solution. Shapiro, Leng and Felsenfeld (Biochemistry, 8:3219-3232, 1969) elucidated the changes in secondary structure associated with the formation of DNA complexes by examining their optical rotation, using a protocol for complexing polylysine to DNA essentially different to that of annealing both components in a step-down salt dialysis. they directly mixed polylysine and DNA in a high salt solvent (1 M NaCl), which resulted in the formation of "soluble` complex. A high degree of turbidity is associated with the complex in solution, indicating aggregation of the components. Aggregation was occurring in the samples used to determine the optical rotatory properties of the complex since the circular dichroism spectra approached the baseline asymptotically at wavelengths in the range of 320 to 360 nm. The anomalous spectrum was always associated with turbidity. We have inferred that the optical activity changes arose from the formation of higher order molecular complexes upon aggregation.
DNA complexes obtained under the experimental conditions described above have a median sedimentation coefficient varying between about 5000 and 10000 units. The average particle had a diameter of 340 nm, (calculated using information provided by light scattering) and the particles had an average dry mass corresponding to about 70 nucleic acid/polypeptide molecular units. The information provided by these studies, while not absolutely quantitative, delineates the structural changes that DNA undergoes after binding to a basic polypeptide.
Several aspects of the structure of DNA-polybase complexes in solution have been investigated (Haynes, Garrett and Gratzer, Biochemistry, 9:4410-4416, 1970). Electron microscopy confirmed the ordered nature of the complexes described by Shapiro et al; DNA structures formed as doughnut-shaped toroids, with an external diameter of 300 nm. The C.D. and electron microscopic features of DNA-poly-base complexes correspond to structural factors residing in the Watson-Crick DNA helix, since single-stranded polynucleotides-polybase complexes i.e. rRNA, po.y(A), poly (U), etc.) do not show anomalous optical activity. Also, ordered structures can be detected in the electron microscope. In order to clarify whether a change in base tilt and/or helix pitch could be observed in the complexes, the X-ray diffraction pattern of the complexes was determined. The double helix is in the normal B form obtained for free DNA in aqueous solutions; no obvious transitions were found to the C or A forms of DNA, suggesting the existence of a different structural form when the DNA is complexed to basic polypeptides in solution. There is also an association of DNA-polybase complexes which involves direct pairing of charges, as shown by the progressive displacement of counter-ions in DNA-polylysine complexes as the salt concentration is decreased. Any strong interaction of the charged amino group with a base is therefore very improbable. Thus, the anomalous rotatory strength of DNA in solution arises from chiral packing, the kind of phenomenon associated with the appearance of a large periodicity in the asymmetric packing of molecules in the same plane.
Lerman et al., Proc. Nat. Acad. Sci. USA, 68:1986-90 (1971) report that when a dilute solution of phage DNA is mixed with a sufficiently high concentration of a simple neutral polymer (polyethylene oxide) in the presence of high NaCl (a simulated intracellular environment), the phage DNA molecules collapse into particles approaching the compactness of the contents of phage heads. The structure of DNA complexes was resolved by Gosule and Schellman (Nature 259: 333-335, 1976). Their publication along with a more detailed report (Gosule, L., Chattoraj. D. K., and Schellman. J., Advances in Polyamine Research 1: 201-215, 1978), showed that the compaction of DNA (in a very dilute solution) by basic polypeptides (spermine and spermidine), under the conditions first described by Li, Biopolymers, 12:287 (1973), resulted in toroid structures. The complexes generated by Gosule and Schellman had an unimolecular structure consisting of a single DNA unit of phage DNA compacted to a maximum radius of about 50 nm. The authors note that polyamines are known to exist in bacterial cells. DNA compaction is also discussed by Laemmli, PNAS 72:4288-92 (1975) and Post and Zimm, Biopolymers, 21:2123-32 (1982).
All references cited in this specification are hereby incorporated by reference. No admission is made that any reference constitutes prior art. The discussion of the references states what their authors assert and applicants reserve the right to challenge the accuracy and pertinency of the cited documents.